Mechanistic and structural characterisation of HydG catalysed L-tyrosine cleavage

Abstract

The S-adenosyl-L-methionine (AdoMet) dependent enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase cofactor assembly. It catalyses L-tyrosine cleavage to yield the cofactor cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve the understanding of the roles of each of these iron?sulfur clusters in catalysis, HydG variants lacking either the N- or C-terminal cluster have been generated and examined using spectroscopic and kinetic methods. Quantification of coordinated iron, UV?Vis spectroscopy and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant which cannot coordinate the radical AdoMet cluster was used to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (?CTD) harbouring only the N-terminal cluster demonstrates that both clusters have similar UV?Vis and EPR spectral properties, but that AdoMet binding and cleavage can only occur at the N-terminal radical AdoMet cluster. The latter observation was also confirmed by initial structural characterisation of a thermostable Thermoanaerobacter italicus HydG. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, Michaelis?Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ?CTD Clostridium acetobutylicum HydG were determined and demonstrate that these C-terminal modifications do not strongly affect the affinity for AdoMet, but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. A definite tyrosine binding site could unfortunately not be established in the Thermoanaerobacter italicus HydG crystal structure. Further kinetic characterisation of the above Clostridium acetobutylicum HydG mutants suggests that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine derived intermediate to cyanide and CO.

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ORCID for Peter L. Roach: orcid.org/0000-0001-9880-2877

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